TDG is a pig-specific epigenetic regulator with insensitivity to H3K9 and H3K27 demethylation in nuclear transfer embryos

Summary Pig cloning by somatic cell nuclear transfer (SCNT) frequently undergoes incomplete epigenetic remodeling during the maternal-to-zygotic transition, which leads to a significant embryonic loss before implantation. Here, we generated the first genome-wide landscapes of histone methylation in pig SCNT embryos. Excessive H3K9me3 and H3K27me3, but not H3K4me3, were observed in the genomic regions with unfaithful embryonic genome activation and donor-cell-specific gene silencing. A combination of H3K9 demethylase KDM4A and GSK126, an inhibitor of H3K27me3 writer, were able to remove these epigenetic barriers and restore the global transcriptome in SCNT embryos. More importantly, thymine DNA glycosylase (TDG) was defined as a pig-specific epigenetic regulator for nuclear reprogramming, which was not reactivated by H3K9me3 and H3K27me3 removal. Both combined treatment and transient TDG overexpression promoted DNA demethylation and enhanced the blastocyst-forming rates of SCNT embryos, thus offering valuable methods to increase the cloning efficiency of genome-edited pigs for agricultural and biomedical purposes.


INTRODUCTION
Pig cloning by somatic cell nuclear transfer (SCNT) holds great promise for agriculture and biomedicine and has been widely employed to produce genome-edited pigs for breed improvement (Xu et al., 2020a;Zheng et al., 2017), human disease research (Yan et al., 2018;Zhang et al., 2018), and organ donation (Niu et al., 2017;Yue et al., 2020). For example, by deleting CD163 and pAPN in donor cells, cloned pigs exhibited resistance to porcine viruses such as PRRSV, TGEV, and PDCoV (Xu et al., 2020a). In addition, PERVKO$3KO$9TG recloned pigs were created for safe and efficient xenotransplantation, which carries 3KO to eliminate xenoantigens, 9TG to enhance immunological compatibility to humans, and PERVKO to prevent viral transmission (Yue et al., 2020). However, despite tremendous efforts over the last two decades, such as modulating embryonic epigenetic modifications  and knocking out XIST in donor cells (Ruan et al., 2018), the birth of full-term cloned pigs remains inefficient (< 3%). Notably, SCNT embryos still exhibit a developmental block during the maternal-to-zygotic transition (MZT), which is considered the consequence of incomplete epigenetic reprogramming in somatic genomes.
In mouse cloning, this incomplete reprogramming has been successfully rectified. Histone 3 lysine 9 trimethyla-tion (H3K9me3) was the first confirmed epigenetic barrier to prevent MZT. Overexpression with H3K9 demethylases in SCNT embryos, such as lysine-specific demethylase 4D (KDM4D) and KDM4B, was able to improve embryonic genome activation (EGA) and the blastocyst rate (90%) (Liu et al., 2016;Matoba et al., 2014). Another repressive mark, H3K27me3, turned out to be more complicated. Injection of either KDM6A mRNA or KDM6B small interfering RNA reduced H3K27me3 enrichments in two-cell embryos and promoted blastocyst formation . A previous report also demonstrated that H3K4me3 impeded Xenopus nuclear reprogramming by maintaining the transcriptional memory of somatic cells (Hormanseder et al., 2017). However, anomalous H3K4me3 might not lead to the EGA arrest of mouse SCNT embryos (Liu et al., 2016). Before histone methylation resetting, DNA hypermethylation was identified in mouse cloning without an effective rescue approach (Tsuji et al., 2009;Yang et al., 2007). Recently, knocking down of DNA methyltransferase was determined to improve mouse cloned EGA and the blastocyst rate, and this rate was even higher (> 95%) when combined with histone demethylase overexpression . All these studies inspired us to investigate epigenetic barriers and defective factors during pig cloned MZT, which previously have not been comprehensively elucidated.
In the present study, we combined RNA sequencing (RNA-seq), ultra-low-input native chromatin immunoprecipitation sequencing (ULI-NChIP-seq), and wholegenome bisulfite sequencing (WGBS) to investigate histone modification and DNA methylation on the donor genome in pig SCNT embryos. More importantly, insufficient activation of thymine DNA glycosylase (TDG), a DNA demethylation regulator specifically expressed during the pig MZT, was defined as a novel barrier for nuclear reprogramming. Taken together, our study reveals a unique interplay between histone modification and DNA methylation in pig nuclear reprogramming and provides effective methods to overcome multiple epigenetic barriers in this process.

Identification of reprogramming-resistant genes and regions in pig SCNT embryos
Large-scale analyses of the embryonic transcriptomes have reported different MZT timing between in vivo-fertilized (IVO) embryos (four-cell stage) and SCNT embryos (eightcell stage) (Cao et al., 2014a;He et al., 2019). Here, we investigated the RNA-seq profiles of two-to eight-cell IVO embryos (IVO2c, IVO4c, IVO8c), four-to eight-cell SCNT embryos (SCNT4c, SCNT8c), and the donor pig fetal fibroblasts (PFF) (Table S1). Both hierarchical clustering and principal component analysis (PCA) (Figures S1A and S1B) indicated that the transcriptional patterns of SCNT8c, but not SCNT4c, were close to IVO4c, reconfirming the MZT delay in SCNT embryos. Nevertheless, 93.4% of SCNT embryos displayed a developmental block at the four-cell stage when treated with transcriptional inhibitor a-amanitin (Table S2), indicating that the newly synthesized RNA of SCNT4c is critical for the subsequent cleavage. Thus, we decided to analyze the transcriptional difference between IVO and SCNT embryos at the same four-cell stage.
EGA-OFF regions are enriched for H3K9me3 and H3K27me3 in SCNT embryos Having ascertained reprogramming-resistant genes and regions in SCNT4c, we then asked whether their anomalous expression patterns are stabilized by epigenetic modifications from donor cells. Therefore, we performed ULI-NChIP-seq on PFF and SCNT4c with 200 cells, and generated the genomewide maps of H3K4me3, H3K9me3 and H3K27me3 (Table  S1). Strikingly, compared with the chromatin immunoprecipitation sequencing (ChIP-seq) using 10e7 PFF (Gao et al., 2019), over 70% of H3K4me3 peaks were detected in our low-input ChIP-seq data ( Figures S2A and S2B). The correlation coefficients between two replicates ranged from 0.85 to 0.98 ( Figure S2C), indicating the high reproducibility of this procedure.
We first assessed the histone methylation on EGA-ON/ OFF regions. As shown in Figure 1C, the ChIP-seq intensities of all three marks in PFF were obviously higher on EGA-OFF regions compared with those on EGA-ON regions. Notably, EGA-OFF regions still preserved H3K9 and H3K27 hypermethylation in SCNT4c, suggesting that these two modifications are pre-existing epigenetic barriers for cloned EGA. A representative region shows the H3K9me3 and H3K27me3 depositions in PFF and SCNT4c with unfaithful gene activation in SCNT4c ( Figure 1D). Simultaneously, EGA-OFF regions were deficiently (legend continued on next page) enriched for H3K4me3 in SCNT4c ( Figure 1C), suggesting that H3K4 hypomethylation is also responsible for EGA failure. We next determined the chromatin structures of EGA-ON/OFF regions by analyzing the distributions of protein-coding genes and repetitive sequences. As expected, EGA-OFF regions contained relatively few genes compared with EGA-ON regions, and they were enriched for repetitive sequences such as LINE and LTR ( Figures S2D and  S2E). These findings indicate that EGA-OFF regions are generally located in heterochromatin regions, which prevents transcriptional activation of the donor genome.
PFF-ON genes and regions are not involved in excessive H3K4me3 in SCNT embryos By further assessing the histone methylation on PFF-ON/ OFF regions, no difference in H3K4me3 was detected among these regions in PFF, and its enrichments were even lower on PFF-ON regions in SCNT4c ( Figure 1E). Moreover, except for H3K27me3 in PFF between partially PFF-OFF and PFF-ON regions, H3K9me3 and H3K27me3 levels in PFF and SCNT4c were significantly higher on PFF-ON regions than those on PFF-OFF regions ( Figure 1E), an example of which is shown in Figure 1F. Since H3K9me3 and H3K27me3 are associated with gene repression (Dambacher et al., 2010), we thus wondered whether transcriptional activation still occurred in these H3K9me3/ H3K27me3-marked regions. Perhaps PFF-ON regions also possess heterochromatin features to maintain the leaky expression from PFF (Saksouk et al., 2015;Vanrobays et al., 2017), because we confirmed that PFF-ON regions were also relatively gene-poor regions enriched with LINE and LTR ( Figures S2D and S2E).
Recently, donor-cell-specific genes expressed in Xenopus SCNT embryos have been reported to exhibit H3K4me3 hypermethylation on their transcription start sites (TSSs) (Hormanseder et al., 2017). However, PFF-ON genes in pigs exhibited the lowest H3K4me3 enrichments around their TSSs (±5 kb) in PFF and SCNT4c when compared with PFF-OFF genes, while EGA-OFF genes only exhibited relatively low H3K4me3 enrichments in PFF when compared with fully EGA-ON genes (Figures 2A-2D). Meanwhile, high levels of H3K9me3 and H3K27me3 were observed around the TSSs of EGA-OFF/PFF-ON genes (Figures 2A-2D), consistent with the results of EGA-OFF/ PFF-ON regions. Taking these data together, we propose that PFF transcriptional profiles preserved in SCNT4c are stabilized by H3K9me3 and H3K27me3, but not H3K4me3.
KDM4A overexpression and GSK126 incubation improve SCNT developmental potential To overcome aberrant H3K9 and H3K27 methylation on EGA-OFF/PFF-ON regions, we injected 1,000 ng/mL mRNA encoding the pig histone demethylases, KDM4A and KDM6A, into one-cell SCNT embryos ( Figure S3A and Table S6). Global histone modifications were then determined by immunofluorescence in SCNT4c (Figures S3B and S3C). In the control group, 100% and 65.2% of embryos were positively stained with H3K9me3 and H3K27me3, respectively. These percentages were decreased to 5%-10% when embryos were injected with KDM4A and KDM6A individually or in combination ( Figure S3C). Nonetheless, compared with the controls (16.6%), KDM6A injection showed no improvement on blastocyst formation (19.5%), and even impaired the beneficial effect of KDM4A (27.5%) when both mRNAs were injected (15.3%) (Figures S3D and S3E; Table S2). Thus, we decided to find another approach to reduce H3K27me3.
Suppression of EZH2 activity, the methyltransferase responsible for establishing H3K27me3, also contributed to nuclear reprogramming (Xie et al., 2016;Zhou et al., 2019). Hence, an EZH2-specific inhibitor, GSK126, was used during cloned MZT ( Figure 3A). As shown in Figures  3B and 3C, the majority of embryos (85.8%) exhibited no H3K27me3 staining when they were treated with 0.1 mM GSK126 for 48 h. More importantly, comparable blastocyst rates were observed in the GSK126-treated (24.7%) and KDM4A-injected groups (26.8%), and this rate was even higher when combining these two methods (33.5%) (Figures 3D and 3E; Table S2). Blastocyst quality was then evaluated by SOX2 immunostaining and TUNEL assay. The results revealed a significant increase of total cell number and inner cell mass cell number in the experimental group, while the number of apoptotic cells showed no decline (Figures S3F and S3G). Furthermore, the experimental group also showed an increased outgrowth colony-forming number with alkaline phosphatase-positive staining (Figures 3F and 3G), suggesting its improved competence for post-implantation development.
Combination of KDM4A and GSK126 restores the transcriptome of SCNT embryos To determine the effects of KDM4A + GSK126 treatment, we performed RNA-seq of combined-treated SCNT4c (SCNT4c-KG). The hierarchical clustering and PCA analysis results revealed that the transcriptional patterns of SCNT4c-KG were (C and E) ChIP-seq intensity of H3K4me3, H3K9me3, and H3K27me3 are shown within EGA-ON/OFF (C) and PFF-ON/OFF (E) regions, and their 100-kb flanking regions. Read counts are normalized by input, total mapped reads, and region length. (D and F) Genome browser view of H3K4me3, H3K9me3, and H3K27me3 enrichments and transcriptional levels in the representative EGA-OFF (D) and PFF-ON (F) regions. See also Figure S2; Tables S1 and S4.  Figure 4G displays the representative genome browser view of two candidate reprogramming-resistant regions containing several restored genes in genes. *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant; two-tailed Student's t test. Reads counts are normalized by input and total mapped reads. See also Figure S1 and Table S3.  (legend continued on next page) SCNT4c-KG. We also analyzed the expression of six EGA-OFF genes ( Figures S5A and S5B) via qPCR, and found that KDM4A + GSK126 treatment successfully upregulated KLF17, PNRC1, SUPT4H1, TFAP2C, and ZSCAN4 ( Figure S5C and Table S6), partially confirming our RNA-seq results ( Figure S5B).
To precisely analyze the DNA methylation changes among PFF, SCNT4c, and SCNT4c-TDG, we performed post-bisulfite adapter tagging sequencing (PBAT-seq), an ultra-low-input WGBS method, to generate their DNA methylation maps on a genome-wide scale. First, when compared with EGA-ON/PFF-OFF regions, DNA methylation levels were higher in EGA-OFF/PFF-ON regions in SCNT4c ( Figure 7A). This confirms that DNA methylation exhibits the same reprogramming-resistant feature as H3K9me3 and H3K27me3 in SCNT4c. Second, the DNA methylation levels of EGA-OFF/PFF-ON regions in SCNT4c-TDG were significantly lower than those in PFF and SCNT4c, indicating an effective DNA demethylation achieved by transient TDG expression ( Figure 7B). DNA demethylation by TDG could also be observed at EGA-OFF/ PFF-ON gene promoters, no matter whether these promoters contained a CpG island or not ( Figure 7C). Third, as examples, the DNA methylation levels of satellite DNA, candidate EGA-OFF gene promoters (TFAP2C, SUPT4H1, KLF17), and PFF-ON gene promoters (UBC, USP5, TRIM8) were examined by bisulfite sequencing PCR (Table S6), and their downtrends of DNA methylation were correlated with their transcriptional restorations in SCNT4c-TDG (Figures S5C and S6A-S6C; Tables S3 and  S5). Fourth, we determined the transcriptional profile of SCNT4c-TDG, which was also close to IVO4c, like SCNT4c-KG ( Figure 7D). Moreover, we found that most   Tables S3 and S4), suggesting that the detailed gene network regulated by H3K9me3, H3K27me3, and DNA methylation are highly similar during pig cloned EGA.

DISCUSSION
In fertilized embryos, histone methylation and DNA methylation undergo a widespread decrease at the pre-im-plantation stage in mouse Liu et al., 2004;Xu et al., 2020b), human (Xu et al., 2020b;Zhu et al., 2018), cattle Zhou et al., 2019Zhou et al., , 2020, and pig (Cao et al., 2014b(Cao et al., , 2015. By contrast, SCNT embryos frequently exhibit abnormal remodeling of these modifications on the donor genome, which is believed to be the primary reason for the significant embryonic losses during cleavage. In this study, we demonstrated that H3K9me3 and H3K27me3, but not H3K4me3, were epigenetic barriers to prevent EGA initiation and somatic cell memory silencing during pig cloned MZT. Moreover, DNA  (legend continued on next page) Stem Cell Reports j Vol. 16 j 2674-2689 j November 9, 2021 2683 methylation was reduced along with H3K9me3 and H3K27me3 removal, whereas DNA demethylation could also be achieved by overexpressing a pig-specific epigenetic regulator, TDG ( Figure 7E).
H3K9me3 and H3K27me3 have been verified as conserved reprogramming barriers among different species. In pig cloning, aberrant H3K9me3 and H3K27me3 have been observed at the one-to two-cell stage via immunofluorescence, while their detrimental effects to MZT remain unknown (Ruan et al., 2018;Xie et al., 2016). By using ULI-NChIP-seq, we showed for the first time that both H3K9me3 and H3K27me3 still existed during the MZT. More precisely, they were deposited on the genomic regions with unfaithful transcriptional patterns, namely EGA-OFF and PFF-ON regions. By contrast, EGA-OFF regions in mouse and human SCNT embryos were only enriched for H3K9me3, but not H3K27me3, and the information on PFF-ON regions was not mentioned (Chung et al., 2015;Matoba et al., 2014). Additionally, most studies utilized the corresponding demethylases to overcome these barriers, such as KDM4A for H3K9me3 in mouse and human (Chung et al., 2015), and KDM6A for H3K27me3 in mouse and cattle Zhou et al., 2019). Our results confirmed their demethylation functions in pig SCNT embryos, but the blastocyst rate was not elevated by KDM6A, regardless of mRNA concentrations (Table S2). Previous studies have individually utilized KDM4A and GSK126 to increase the pig SCNT blastocyst rate (Ruan et al., 2018;Xie et al., 2016). Here, we combined these two methods with a longer GSK126 treatment time (up to 48 h) to further facilitate blastocyst formation, which might be due to an obvious reduction in cloned MZT defect.
In addition to EGA failure, persistent expression of donor-cell-specific genes, namely PFF-ON genes, also derives from incomplete reprogramming. Currently, excessive H3K4me3 has been detected in cattle eight-cell SCNT embryos  and on the TSSs of PFF-ON genes in Xenopus donor cells (Hormanseder et al., 2017). Here, we found that PFF-ON regions or the TSSs of PFF-ON genes in pig cloning were enriched for H3K9me3 and H3K27me3, but not H3K4me3. Remarkably, most PFF-ON genes and regions were silenced through H3K9me3 and H3K27me3 removal. We also noticed that PFF-ON genes in Xenopus and cattle SCNT embryos were involved in donor cell identity, such as endoderm-and fibroblast-specific genes (Hormanseder et al., 2017;Zhou et al., 2020). In contrast, only nine PFF-ON genes were fibroblast-specific genes when we compared our PFF-ON genes with a list of 563 marker genes for mouse fibroblasts (Table S3) (Kim et al., 2010). Therefore, although SCNT embryos harbor PFF-ON genes in many species, the composition of PFF-ON genes and how they maintain their transcriptions are likely different between pig and other species. Another difference between our work and previous studies is the effect of H3K4me3 removal for reprogramming. H3K4 demethylation has been proved to promote development in mouse, cattle, and Xenopus cloning (Hormanseder et al., 2017;Liu et al., 2016;Zhou et al., 2020). Accordingly, we injected KDM5A mRNA (Table S6) and tested its H3K4 demethylase function in four-cell embryos ( Figures S7F and S7G). However, KDM5A overexpression could not rescue the EGA defect and the poor developmental phenotype of pig SCNT embryos ( Figure S5C and Table S2).
Soon after the establishment of the SCNT technology, DNA hypermethylation was the first determined epigenetic barrier. Deficient 5mC and 5hmC demethylation has been found in pig and cattle SCNT embryos by immunostaining (Cao et al., 2014b;Zhou et al., 2020). Most studies utilized 5-aza-2-deoxycytidine (5AZA), a DNA methyltransferase inhibitor, to deplete 5mC. However, improvements could be observed in human and pig (Huan et al., 2013;Sun et al., 2012) but not in mouse and cow (Ding et al., 2008;Tsuji et al., 2009). In this study, we investigated the interplay between histone modification and DNA methylation, since KDM4A + GSK126 treatment decreased the numbers of embryos with 5mC-and 5hmC-positive staining. Of note, DNMT1 and DNMT3B are downregulated by this treatment (Table S3), similar to the effect of 5AZA. Furthermore, TDG is likely to serve as a rate-limiting factor for DNA demethylation in pig nuclear reprogramming, because its expression not only removes 5caC but also removes the upstream 5mC and 5hmC. In this study, we also combined KDM4A + GSK126 treatment with transient TDG expression to verify whether this triple treatment could raise the efficiency of nuclear reprogramming. Unexpectedly, it showed no more improvement of cloned blastocyst formation (Table S2). We speculate that (E) Immunostaining of 5mC (green), 5hmC (green), 5fC (red), and 5caC (red) in SCNT4c derived from TDG + Dox, combined-treated, and non-treated groups. One of the nuclei in SCNT4c is magnified 5-fold. Scale bars, 50 mm.  the developmental improvement achieved by TDG is mainly related to 5mC demethylation, which is shared by KDM4A + GSK126 treatment. Perhaps 5caC accumulation in SCNT4c-KG may not be a reprogramming barrier for MZT, thus further removing 5caC by TDG may not show a further improvement by triple treatment. Future studies also should elucidate two additional questions: (1) 5mC and 5hmC were comparably enriched in SCNT embryos when compared with PFF, while 5fC and 5caC were reduced in SCNT embryos ( Figures 6F and S7H). What causes this difference? (2) AICDA and SMUG1 process EGA-specific profiles and fail to be activated in SCNT embryos (Table S3). What is the role of 5mC deamination in nuclear reprogramming?
In summary, our study advances the understanding of epigenetic remodeling in pig SCNT embryos on a genomewide scale and provides an effective combination strategy to remove multiple barriers during the MZT. Meanwhile, we defined TDG as a novel and species-specific regulator to potentiate the developmental competence of SCNT embryos. Recently, the H3K9me3 methyltransferase inhibitor chaetocin exhibited a beneficial effect in pig cloning . Therefore, combinational use of chaetocin, GSK126, and 5AZA will require optimization to further improve the cloning efficiency. This work will accelerate the practical use of the SCNT technique for pig model production and contribute to the studies of human disease, xenotransplantation, and molecular breeding in agriculture.

EXPERIMENTAL PROCEDURES
All experimental procedures were approved by the Animal Care Commission of Huazhong Agriculture University, Wuhan, China.

RNA sequencing and data processing
The total RNA of PFF and 30 SCNT embryos were extracted by TRIzol (Invitrogen) or a PicoPure RNA Isolation Kit (Applied Biosystems), respectively. Double-stranded cDNA was then synthesized by a SMARTer Pico PCR cDNA Synthesis Kit (Clontech) and an Advantage 2 PCR Kit (Clontech), and purified by AMPure XP beads (Beckman Coulter). After fragmentation with a Bioruptor Sonication System (Diagenode), cDNA libraries were generated by a VAHTS Universal V6 RNA-seq Library Prep Kit (Vazyme). Paired-end 150-bp sequencing was performed on an Illumina Hi-Seq X Ten platform.

Identification of differentially expressed regions
The expression levels of genomic regions among samples were assessed through a sliding window (size 50 kb, step size 20 kb) made by bedtools, and normalized by RPM. Differentially expressed regions (DERs) in Figure 1 were extracted using the following criteria: EGA-ON regions in IVO4c, FC IVO4c/IVO2c > 5, average RPM in IVO4c > 5; PFF-OFF regions in IVO4c, FC PFF/IVO4c > 5, average RPM in PFF > 5. Both regions were classified into three groups based on the expression differences among IVO4c, PFF, and SCNT4c. We also defined FC SCNT4c-KG/SCNT4c > 2 and FC SCNT4c-TDG/SCNT4c > 2 as EGA-OFF regions reactivated in SCNT4c-KG/TDG, while FC SCNT4c/SCNT4c-KG > 2 and FC SCNT4c/SCNT4c-TDG > 2 were PFF-ON regions silenced in SCNT4c-KG/TDG. Fisher's exact test p values of all DERs were < 0.05.

ULI-NChIP sequencing and data processing
ULI-NChIP was performed as previously described (Brind'Amour et al., 2015). For each immunoprecipitation reaction, 200 PFF or 50 four-cell embryos were added in Nuclear Isolation buffer and MNase Master Mix to digest chromatin for 7 min at 25 C. Chromatin was diluted in Complete Immunoprecipitation buffer, and incubated with 2 mg of antibody-bead complexes (H3K4me3, somatic cell memory silencing. Combining KDM4A overexpression and GSK126 incubation could remove H3K9me3, H3K27me3, and DNA methylation, restore the global transcriptome and repeatome, and provide a double increase for cloned blastocyst formation. Moreover, DNA demethylation and developmental improvement could also be achieved by TDG overexpression. See also Figures S6 and S7; Tables S3 and S4.

Statistical analysis
All experiments were repeated at least three times. All middle lines in the box-whisker plots indicate the median, the edges indicate the 25 th /75 th percentiles, and the whiskers indicate the 2.5 th /97.5 th percentiles. Development rates and qPCR results are presented as the mean ± SD. p values were calculated using two-tailed Student's t test with SPSS Statistics 20 software (IBM). Differences are shown with asterisks indicating *p < 0.05, **p < 0.01, and ***p < 0.001.

Data and code availability
The Gene Expression Omnibus accession numbers for the data reported in this paper are GEO: GSE161527 and GSE139512.

CONFLICTS OF INTERESTS
The authors declare no competing interests.